Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

Abstract

ABSTRACTWe report engineeringNeurospora crassato improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain ofN. crassa(F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes,cre-1andace-1, on cellulase production were investigated. The F5 Δace-1mutant showed no improvement over the wild type. The F5 Δcre-1and F5 Δace-1Δcre-1strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1Δcre-1to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1Δcre-1ΔndvBstrain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1Δcre-1.